| ObjectiveAcute lung injury/Acute respiratory distress syndrome (ALI/ARDS) is an acute progressive respiratory failure of hypoxemia. The pathogenesis of ARDS is complex and unclear. At present, the treatment of ARDS is mainly supportive on the organs, especially the respiratory supporting therapy. In the past two decades, much effort was made on the therapeutics of it. Although the mortality is decreased, it still topped at 40%. So, much attention should be focus on constructing the therapeutic interventions for the pathogenesis of ALI. Cell apoptosis plays an essential role in the progression of ALI/ARDS. The apoptosis of AEC-2 and PVEC is the main factor of the ALI/ARDS because it leads to increased permeability, extravasations of protein rich fluid and interstitial edema, pulmonary atelectasis, increasing resistance of pulmonary vascular and so on. So, blocking the apoptosis of AEC-2 and PVEC may be new strategy in the cure of ALI/ARDS. Both LPS and TNF-αcan induce the apoptosis of AEC and PVEC by activating Caspase-8. FADD-like interleukin-1-βconverting enzyme inhibitory protein(FLIP) is an important apoptosis inhibitor. FLIP has the domain similar with Caspase-8 and could compete with caspase-8 in combining with FADD, inhibit the conformation of DISC and block cell apoptosis. Our preliminary experiments have already clarified that FLIP has an anti-apoptosis cytoprotective effect in cultured AEC and PVEC in the models of Fas-mediated cell apoptosis. To confirm its protective effect in ALI animal models, we made the rats infected with Ad-FLIP. Then we investigated the level of FLIP expressions in the lungs and its anti-apoptosis of AEC-2 and PVEC in lung, evaluated its role in preventing the development of ALI/ARDS. Additionally, we investigated probable mechanisms of FLIP in inhibiting cell apoptosis. This study might provide us crucial experimental evidence on the gene therapeutic strategies of ALI/ARDS.Methods1. Best expression time of Ad-FLIP. Twenty four SD rats were randomly divided into four groups according to the different killing time after Ad-EGFP vector administration, with 6 rats in each. Rats were anesthetized by intraperitoneal injection of pentobarbital (1%, 50mg/kg) and then inhaled Ad-EGFP vector (500ul/kg). The right middle and accessory lobes of the lungs were harvested separately at 24, 48, 72, and 96h after Ad-EGFP administration. The expressions of EGFP in the lungs were observed by fluorescence microscope.2. Treatment of animal model. Fourty eight SD rats were randomly divided into four groups, with 12 rats in each. In treatment group, ALI rat models were established by LPS intraperitoneal injection (5mg/kg) and then inhaled Ad-FLIP vector 4 hours later. In prevention group,the animals were infected with Ad-FLIP vector 48 hours before ALI models were established. Two control groups of treatment and prevention received Ad-EGFP vectors respectively.3. Effects of Ad-FLIP on expression of FLIP in lung and ALI/AIDS. mRNA and protein expressions of FLIP in lungs were investigated by RT-PCR and immunohistochemistry. Evaluate the effects of Ad-FLIP in the role of prevention and treatment of ALI/ARDS. Pathological changes of lung were observed by light microscope. Wet weight/ dry weight ratios (W/D) of lung lobes and Lung permeability index (LPI) were also measured.4. Apoptosis of lung cells. Apoptosis of AEC-2 and PVEC in lungs were observed by transmission electron microscope (TEM). The phenomenon of DNA Ladder in lung tissues was detected by agarose electrophoresis after the total DNA of lungs was extracted. The protein expressions of Bax and Bcl-2 in lungs were investigated by Western Blot and immunohistochemistry respectively.Results1. Inhaling adenovirus vector with EGFP gene can significantly up-regulate the fluorescent expression of EGFP protein in rat lung, especially 48h after administration of Ad-EGFP in vivo.2. With administration of Ad-FLIP in vivo, it was found that pulmonary histopathology improved, the permeability of respiratory capillary membrane significantly reduced,the expressions of FLIP mRNA and protein in lung tissues elevated both in the treatment group and prevention group.3. In treatment group and prevention group, AEC-2 and PVEC apoptosis notablely decreased in lungs and the DNA Ladder was not identified. The protein expression of Bax in lung tissues decreased while Bcl-2 was upregulated. The ratio of Bax/Bcl-2 is cut down.Conclusion The data from our study suggest that inhaling Ad-FLIP can not only successfully infect the rat but also elevate the expression of FLIP in lung. Moreover, pulmonary edema and permeability of respiratory membrane induced by LPS in ALI rat models were attenuated by Ad-FLIP treatment or pretreatment. It was also suggested that Ad-FLIP has effects on ALI/ARDS both in treatment and prevention. Blocking the apoptosis of AEC-2 and PVEC is the crucial mechanism which may involve the regulation of Bax/Bcl-2 ratio. This study provided us a novel and effective way to prevent the progression of ALI/ARDS.
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